DNA southern blot hybridization
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DNA southern blot hybridization
Southern blot hybridization is one of the most commonly used molecular biology techniques to detect Specific DNA sequences using labeled probes.
The DNA southern blot hybridizatin is illustrated bellow:
General DNA southern blot procedures:
1) Extract genomic DNA (or purified double-stranded DNA) from source organism. Digest DNA with a chosen restriction enzyme that can give a specific digestion pattern for further analysis. Run agarose electrophoresis to separate the fragments of digested DNA.
2) Transfer DNA onto membrane (nylone, nitrocellular membranes).
3) Hybridize the membrane with labeled probe (single stranded DNA with sequence complementary to the target DNA). The commonly used probes include oligonucleotide probe, cDNA probe.
4) Wash membrane to remove non-specific binding, develop x-film to reveal the specific hybridization signals.
Online resources for DNA southern blot:
DNA southern blot procedure
Assembly of southern blot hybridization apparatus for transfer
Last update 17-Mar-2004, Rating Good of 2 votes.
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Materials:
• 0.5 M Tris.HCl +1.5M NaCl
• 6xSSC: (133.2 ml)
20 x SSC 40 ml
Sterile Water 93.2 ml
• 6x SSC + 0.5% SDS: (200 ml)
20 x SSC 60 ml
10% SDS 10 ml
• 2x SSC + 0.1% SDS (200 ml)
20 x SSC 20 ml
10% SDS 2 ml
• 6x SSC + 0.5% SDS + 5 x Denhardt (500 ml)
20 x SSC 150 ml
10% SDS 25 ml
100 x Denhardt solution 25 ml
Procedure:
1. Capillary transfer of cDNA into nylone membrane overnight.
2. Neutralize filter in 0.5 M Tris.HCl +1.5M NaCl for 30 min.
3. Dry filter in air. (1 h or so)
4. Fixation under UV-lamp. less than 1 min.
5. (5-10 steps performed in hybridization plastic box) Wash filter with 6x SSC. (Wet filter 2 min)
6. Discard liquid, Wash filter in 6x SSC +0.5% SDS solution for 30 min.
7. Discard liquid, Add 6x SSC+0.5% SDS+5x Denhardt (hybridization buffer) at 50 C for 30 min.
8. Preparing 100 C water bath/ 95 C cycler.
9. Boiling haringsperma (10mg/ml) for 5 min. Snap chill on ice.
10. Discard solution, add 25 ml hybridization buffer + 400 ul haringsperma (10 mg/ml) at 50 C for 30 min. (Prehybridization)
11. Add all labeled human motilin cDNA probe.
12. Incubate hybridization box in water bath of 50 C overnight.
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13. Remove the filter and immediately submerge it in a tray containing 200 ml 2x SSC+ 0.1% SDS in room temperature for 1 hour.
14. Wash in 2 x SSC for 30 min , change fresh 2x SSC for another 30 min.
15. Dry filter with paper towel.
16. Place the damp filter on a sheet of saran wrap and cover the filter. Expose the filter to X-ray film for 1-2 days.
17. X-film:
• -70 C for 1-2 days
• Prewarm in room termperature for 30 min.
• Development 1.5 min
• Acetic acid stop 2 min
• Fixation for 2 min
• Water wash for 5 min.
• Dry
Rating: Excellent!
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What should be the pH of Hybridization buffer?
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I've done southern blotting with 10mcg DNA ,and hybridized with specific probe at 65 degrees.
After 3xsscwith 0.1%SDS , 2x ssc(0.1%SDS),and !x ssc (0.1%SDS) , the count is not decreasing from 400.
an you suggest me the solution for this
Rating: Good
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